A Nonhuman Primate Model of Fragile X Associated Primary Ovarian Insufficiency Abstract Fragile X encompasses a range of genetic conditions, all of which result as a function of changes within the FMR1 gene and abnormal production and/or expression of the FMR1 gene products. The normal allele (5-54 CGG repeats) is stably transmitted to offspring and FMR protein (FMRP) expression is normal. In individuals that carry the pre-mutation sequence (55-200 CGG repeat), the alleles remain unmethylated and are associated with increasing levels of FMR1 transcripts and decreasing levels of FMRP. Females with the Fragile X pre-mutation (FXPM) are at risk of developing FXPOI and early onset menopause, the only non- neurological/psychological condition attributed to the pre-mutation range of CGG repeat lengths. To fully investigate the molecular mechanisms and biological consequences of the human pre-mutation CGG repeat expansion responsible for FXPOI it is necessary to design and characterize appropriate in-vitro and in-vivo model systems. Embryonic stem cells (ESC) are an ideal tool with which to study developmentally regulated cell function due to their pluripotency, unlimited capacity for self-renewal, ability to accept targeted genetic manipulations and undergo directed differentiation. This proposal will seek to create for the first time in-vitro and in-vivo NHP models of FXPOI. Three specific aims will be addressed; (1) Generation of a NHP ESC-based model of FXPOI. Using site-specific transgenesis the human FXPM CGG repeat sequence is introduced into the endogenous FMR1 gene of a currently existing female NHP ESC line. The FXPM transgenic ESC line (ESCPOI) will then be used for the in-vitro differentiation and characterization of FXPM primordial germ cells. (2) Generation of female ESCPOI-chimeric NHP infants. Using the ESCPOI line from Aim 1, in conjunction with optimized techniques for the in-vitro production of ESC-chimeric embryos, we will generate female NHP infants that show expression of the FXPM within ovarian tissue (germline ESCPOI-chimeras). This specific aim represents a proof-of-principle step that will be fundamental to future full-scale expansion of an in-vivo NHP FXPOI model system. (3) For model characterization, ESCPOI expression in both somatic and germline tissues of liveborn infants will be assessed. Physical growth and neurobehavioral assessment in infants up to 6 months of age will be monitored using specialized testing procedures in our state-of-the-art Infant Primate Research Laboratory to determine if infants display any neurological deficits associated with the FXPM. Our proposal is focused on the use of ESC-based technologies in the NHP in an effort to provide renewable, defined and clinically relevant translational in-vitro and in-vivo NHP model systems of FXPOI to the broader Fragile X research community.